The long-term objective of the proposed research is to understand gating mechanisms in K+ channels. K+ channels are critical to the generation of electrical impulses by excitable cells. They have been the subject of intense research, however, a number of key questions remain. One of the reasons why these questions are still open is the limited ability of traditional site-directed mutagenesis to modify the channel structure in a precise way. This approach allows amino acid substitutions that are limited to the set of twenty naturally occurring amino acids. A higher accuracy for modifications of the structural and electronic properties of the protein can be achieved by means of unnatural amino acid mutagenesis. Chemical synthesis is a powerful approach for unnatural amino acid mutagenesis that offers an almost unlimited choice of amino acid side chain and peptide backbone modifications. Such modifications are not possible using traditional site-directed mutagenesis. The goal of this application is to develop general strategies for the chemical synthesis of ion channel proteins and to apply it to investigation on the inactivation process in potassium channels. In my postdoctoral research, 1 proposed to investigate the gating process referred to as "slow inactivation" in the bacterial K+ channel, KcsA. I will pursue two major specific aims: Aim 1) To optimize chemical synthesis methodology for investigating membrane proteins in vitro. The chemical synthesis protocols that will be developed within my postdoctoral training will be useful not only for investigating the KcsA channel but will also find applicability in the chemical synthesis of other important classes of membrane proteins. Aim 2) To investigate the mechanism of KcsA inactivation using chemical synthesis. I propose that a better understanding of the KcsA inactivation may be achieved by using unnatural amino acid mutagenesis. For my research, I will use the chemical synthesis to precisely perturb the selectivity filter of the KcsA channel and its interactions with the surrounding protein. This approach will allow me to evaluate the effect of channel modifications on slow inactivation that will be determined by electrophysiological measurements. My research will help to establish the molecular basis for the process of slow inactivation.